Cryo-electron Microscopy Research Articles

Solution Behavior of Glyco-Copoly(l-Glutamic Acid)s in Dilute Saline Solution.

A small series of copoly(α,l-glutamic acid/dl-allylglycine)s with the same chain length and allylglycine content (∼10 mol %) but different spatial distribution of allylglycine units was synthesized and subsequently glycosylated via thiol-ene chemistry. Dilute aqueous copolypeptide solutions (0.1 wt %, physiological saline) were analyzed by circular dichroism spectroscopy, dynamic light scattering, and cryogenic transmission electron microscopy. The copolypeptides adopted a random coil or α-helix conformation, depending on solution pH, and the glycosylated residues either distorted or enhanced the folding into an α-helix depending on their location and spatial distribution along the chain. However, regardless of their secondary structure and degree of charging, all partially glycosylated copolypeptides self-assembled into 3D spherical structures, supposedly driven by a hydrophilic effect promoting microphase separation into glucose-rich and glutamate-rich domains.

Structural basis of the Meinwald rearrangement catalysed by styrene oxide isomerase.

Membrane-bound styrene oxide isomerase (SOI) catalyses the Meinwald rearrangement-a Lewis-acid-catalysed isomerization of an epoxide to a carbonyl compound-and has been used in single and cascade reactions. However, the structural information that explains its reaction mechanism has remained elusive. Here we determine cryo-electron microscopy (cryo-EM) structures of SOI bound to a single-domain antibody with and without the competitive inhibitor benzylamine, and elucidate the catalytic mechanism using electron paramagnetic resonance spectroscopy, functional assays, biophysical methods and docking experiments. We find ferric haem b bound at the subunit interface of the trimeric enzyme through H58, where Fe(III) acts as the Lewis acid by binding to the epoxide oxygen. Y103 and N64 and a hydrophobic pocket binding the oxygen of the epoxide and the aryl group, respectively, position substrates in a manner that explains the high regio-selectivity and stereo-specificity of SOI. Our findings can support extending the range of epoxide substrates and be used to potentially repurpose SOI for the catalysis of new-to-nature Fe-based chemical reactions.

Selection and characterization of aptamers targeting the Vif-CBFβ-ELOB-ELOC-CUL5 complex.

The viral infectivity factor (Vif) of human immunodeficiency virus 1 forms a complex with host proteins, designated as Vif-CBFβ-ELOB-ELOC-CUL5 (VβBCC), initiating the ubiquitination and subsequent proteasomal degradation of the human antiviral protein APOBEC3G (A3G), thereby negating its antiviral function. While recent cryo-electron microscopy (cryo-EM) studies have implicated RNA molecules in the Vif-A3G interaction that leads to A3G ubiquitination, our findings indicated that the VβBCC complex can also directly impede A3G-mediated DNA deamination, bypassing the proteasomal degradation pathway. Employing the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method, we have identified RNA aptamers with high affinity for the VβBCC complex. These aptamers not only bind to the VβBCC complex but also reinstate A3G's DNA deamination activity by inhibiting the complex's function. Moreover, we delineated the sequences and secondary structures of these aptamers, providing insights into the mechanistic aspects of A3G inhibition by the VβBCC complex. Analysis using selected aptamers will enhance our understanding of the inhibition of A3G by the VβBCC complex, offering potential avenues for therapeutic intervention.

Microfibril-associated glycoprotein 4 forms octamers that mediate interactions with elastogenic proteins and cells

Microfibril-associated glycoprotein 4 (MFAP4) is a 36-kDa extracellular matrix glycoprotein with critical roles in organ fibrosis, chronic obstructive pulmonary disease, and cardiovascular disorders, including aortic aneurysms. MFAP4 multimerises and interacts with elastogenic proteins, including fibrillin-1 and tropoelastin, and with cells via integrins. Structural details of MFAP4 and its potential interfaces for these interactions are unknown. Here, we present a cryo-electron microscopy structure of human MFAP4. In the presence of calcium, MFAP4 assembles as an octamer, where two sets of homodimers constitute the top and bottom halves of each octamer. Each homodimer is linked together by an intermolecular disulphide bond. A C34S missense mutation prevents disulphide-bond formation between monomers but does not prevent octamer assembly. The atomic model, built into the 3.55 Å cryo-EM map, suggests that salt-bridge interactions mediate homodimer assembly, while non-polar residues form the interface between octamer halves. In the absence of calcium, an MFAP4 octamer dissociates into two tetramers. Binding studies with fibrillin-1, tropoelastin, LTBP4, and small fibulins show that MFAP4 has multiple surfaces for protein-protein interactions, most of which depend upon MFAP4 octamer assembly. The C34S mutation does not affect these protein interactions or cell interactions. MFAP4 assemblies with fibrillin-1 abrogate MFAP4 interactions with cells.

Structural basis of negative regulation of CRISPR-Cas7-11 by TPR-CHAT

CRISPR‒Cas7-11 is a Type III-E CRISPR-associated nuclease that functions as a potent RNA editing tool. Tetratrico-peptide repeat fused with Cas/HEF1-associated signal transducer (TPR-CHAT) acts as a regulatory protein that interacts with CRISPR RNA (crRNA)-bound Cas7-11 to form a CRISPR-guided caspase complex (Craspase). However, the precise modulation of Cas7-11’s nuclease activity by TPR-CHAT to enhance its utility requires further study. Here, we report cryo-electron microscopy (cryo-EM) structures of Desulfonema ishimotonii (Di) Cas7-11-crRNA, complexed with or without the full length or the N-terminus of TPR-CHAT. These structures unveil the molecular features of the Craspase complex. Structural analysis, combined with in vitro nuclease assay and electrophoretic mobility shift assay, reveals that DiTPR-CHAT negatively regulates the activity of DiCas7-11 by preventing target RNA from binding through the N-terminal 65 amino acids of DiTPR-CHAT (DiTPR-CHATNTD). Our work demonstrates that DiTPR-CHATNTD can function as a small unit of DiCas7-11 regulator, potentially enabling safe applications to prevent overcutting and off-target effects of the CRISPR‒Cas7-11 system.

Enhanced potency of an IgM-like nanobody targeting conserved epitope in SARS-CoV-2 spike N-terminal domain

Almost all the neutralizing antibodies targeting the receptor-binding domain (RBD) of spike (S) protein show weakened or lost efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged or emerging variants, such as Omicron and its sub-variants. This suggests that highly conserved epitopes are crucial for the development of neutralizing antibodies. Here, we present one nanobody, N235, displaying broad neutralization against the SARS-CoV-2 prototype and multiple variants, including the newly emerged Omicron and its sub-variants. Cryo-electron microscopy demonstrates N235 binds a novel, conserved, cryptic epitope in the N-terminal domain (NTD) of the S protein, which interferes with the RBD in the neighboring S protein. The neutralization mechanism interpreted via flow cytometry and Western blot shows that N235 appears to induce the S1 subunit shedding from the trimeric S complex. Furthermore, a nano-IgM construct (MN235), engineered by fusing N235 with the human IgM Fc region, displays prevention via inducing S1 shedding and cross-linking virus particles. Compared to N235, MN235 exhibits varied enhancement in neutralization against pseudotyped and authentic viruses in vitro. The intranasal administration of MN235 in low doses can effectively prevent the infection of Omicron sub-variant BA.1 and XBB in vivo, suggesting that it can be developed as a promising prophylactic antibody to cope with the ongoing and future infection.

Structural insights into drug transport by an aquaglyceroporin

Pentamidine and melarsoprol are primary drugs used to treat the lethal human sleeping sickness caused by the parasite Trypanosoma brucei. Cross-resistance to these two drugs has recently been linked to aquaglyceroporin 2 of the trypanosome (TbAQP2). TbAQP2 is the first member of the aquaporin family described as capable of drug transport; however, the underlying mechanism remains unclear. Here, we present cryo-electron microscopy structures of TbAQP2 bound to pentamidine or melarsoprol. Our structural studies, together with the molecular dynamic simulations, reveal the mechanisms shaping substrate specificity and drug permeation. Multiple amino acids in TbAQP2, near the extracellular entrance and inside the pore, create an expanded conducting tunnel, sterically and energetically allowing the permeation of pentamidine and melarsoprol. Our study elucidates the mechanism of drug transport by TbAQP2, providing valuable insights to inform the design of drugs against trypanosomiasis.

Program VUE: analysing distributions of cryo-EM projections using uniform spherical grids

Three-dimensional cryo electron microscopy reconstructions are obtained by extracting information from a large number of projections of the object. These projections correspond to different `views' or `orientations', i.e. directions in which these projections show the reconstructed object. Uneven distribution of these views and the presence of dominating preferred orientations may distort the reconstructed spatial images. This work describes the program VUE (views on uniform grids for cryo electron microscopy), designed to study such distributions. Its algorithms, based on uniform virtual grids on a sphere, allow an easy calculation and accurate quantitative analysis of the frequency distribution of the views. The key computational element is the Lambert azimuthal equal-area projection of a spherical uniform grid onto a disc. This projection keeps the surface area constant and represents the frequency distribution with no visual bias. Since it has multiple tunable parameters, the program is easily adaptable to individual needs, and to the features of a particular project or of the figure to be produced. It can help identify problems related to an uneven distribution of views. Optionally, it can modify the list of projections, distributing the views more uniformly. The program can also be used as a teaching tool.

Point mutation in a virus-like capsid drives symmetry reduction to form tetrahedral cages

Protein capsids are a widespread form of compartmentalization in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry maximizes the internal volume that can be enclosed within. Despite the strong preference for icosahedral symmetry, we show that simple point mutations in a virus-like capsid can drive the assembly of unique symmetry-reduced structures. Starting with the encapsulin from Myxococcus xanthus , a 180-mer bacterial capsid that adopts the well-studied viral HK97 fold, we use mass photometry and native charge detection mass spectrometry to identify a triple histidine point mutant that forms smaller dimorphic assemblies. Using cryoelectron microscopy, we determine the structures of a precedented 60-mer icosahedral assembly and an unexpected 36-mer tetrahedron that features significant geometric rearrangements around a new interaction surface between capsid protomers. We subsequently find that the tetrahedral assembly can be generated by triple-point mutation to various amino acids and that even a single histidine point mutation is sufficient to form tetrahedra. These findings represent a unique example of tetrahedral geometry when surveying all characterized encapsulins, HK97-like capsids, or indeed any virus-derived capsids reported in the Protein Data Bank, revealing the surprising plasticity of capsid self-assembly that can be accessed through minimal changes in the protein sequence.

STORM Super-Resolution Visualization of Self-Assembled γPFD Chaperone Ultrastructures in Methanocaldococcus jannaschii.

Gamma-prefoldin (γPFD), a unique chaperone found in the extremely thermophilic methanogen Methanocaldococcus jannaschii, self-assembles into filaments in vitro, which so far have been observed using transmission electron microscopy and cryo-electron microscopy. Utilizing three-dimensional stochastic optical reconstruction microscopy (3D-STORM), here we achieve ∼20 nm resolution by precisely locating individual fluorescent molecules, hence resolving γPFD ultrastructure both in vitro and in vivo. Through CF647 NHS ester labeling, we first demonstrate the accurate visualization of filaments and bundles with purified γPFD. Next, by implementing immunofluorescence labeling after creating a 3xFLAG-tagged γPFD strain, we successfully visualize γPFD in M. jannaschii cells. Through 3D-STORM and two-color STORM imaging with DNA, we show the widespread distribution of filamentous γPFD structures within the cell. These findings provide valuable insights into the structure and localization of γPFD, opening up possibilities for studying intriguing nanoscale components not only in archaea but also in other microorganisms.

Structural and Computational Insights into Dynamics and Intermediate States of Orexin 2 Receptor Signaling.

Orexin 2 receptor (OX2R) is a G protein-coupled receptor (GPCR) whose activation is crucial to regulation of the sleep-wake cycle. Recently, inactive and active state structures were determined from X-ray crystallography and cryo-electron microscopy single particle analysis, and the activation mechanisms have been discussed based on these static data. GPCRs have multiscale intermediate states during activation, and insights into these dynamics and intermediate states may aid the precise control of intracellular signaling by ligands in drug discovery. Molecular dynamics (MD) simulations are used to investigate dynamics induced in response to thermal perturbations, such as structural fluctuations of main and side chains. In this study, we proposed collective motions of the TM domain during activation by performing 30 independent microsecond-scale MD simulations for various OX2R systems and applying relaxation mode analysis. The analysis results suggested that TM3 had a vertical structural movement relative to the membrane surface during activation. In addition, we extracted three characteristic amino acid residues on TM3, i.e., Q1343.32, V1423.40, and R1523.50, which exhibited large conformational fluctuations. We quantitatively evaluated the changes in their equilibrium during activation in relation to the movement of TM3. We also discuss the regulation of ligand binding recognition and intracellular signal selectivity by changes in the equilibrium of Q1343.32 and R1523.50, respectively, according to MD simulations and GPCR database. Additionally, the OX2R-Gi signaling complex is stabilized in the conformation resembling a non-canonical (NC) state, which was previously proposed as an intermediate state during activation of neurotensin 1 receptor. Insights into the dynamics and intermediate states during activation gained from this study may be useful for developing biased agonists for OX2R.

Cryo-EM combined with image deconvolution to determine ZIF-8 crystal structure

Abstract Metal-organic frameworks (MOFs) are crystalline porous materials with tunable properties, exhibiting great potential in gas adsorption, separation and catalysis.[1, 2] It is challenging to visualize MOFs with transmission electron microscopy (TEM) due to their inherent instability under electron beam irradiation. Here, we employ cryo-electron microscopy (cryo-EM) to capture images of MOF ZIF-8, revealing inverted-space structural information at a resolution of up to about 1.7 Å and enhancing its critical electron dose to around 20 e-/Å2. In addition, it is confirmed by electron-beam irradiation experiments that the high voltage could effectively mitigate the radiolysis, and the structure of ZIF-8 is more stable along the [100] direction under electron beam irradiation. Meanwhile, since the high-resolution electron microscope images is modulated by contrast transfer function (CTF) and it is difficult to determine the positions corresponding to the atomic columns directly from the images. We employ image deconvolution to eliminate the impact of CTF and obtain the structural images of ZIF-8. As a result, the heavy atom Zn and the organic imidazole ring within the organic framework can be distinguished from structural images.

Flash melting amorphous ice.

Water can be vitrified if it is cooled at high rates, which makes it possible to outrun crystallization in so-called no man's land, a range of deeply supercooled temperatures where water crystallizes rapidly. Here, we study the reverse process in pure water samples by flash melting amorphous ice with microsecond laser pulses. Time-resolved electron diffraction reveals that the sample transiently crystallizes despite a heating rate of more than 5 × 106 K/s, even though under the same conditions, vitrification can be achieved with a similar cooling rate of 107 K/s. Moreover, we observe different crystallization kinetics for amorphous solid water and hyperquenched glassy water. These experiments open up new avenues for elucidating the crystallization mechanism of water and studying its dynamics in no man's land. They also add important insights into the laser melting and revitrification processes that are integral to the emerging field of microsecond time-resolved cryo-electron microscopy.

Environmentally friendly fluorine-free fire extinguishing agent based on the synergistic effect of silicone, hydrocarbon surfactants and foam stabilizers

The environmentally friendly fluorine-free fire extinguishing agent based on the silicone surfactant was developed in order to solve the environmental hazards and bio-accumulative of traditional aqueous film-forming foam (AFFF). The carboxyl modified polyether polysiloxane surfactant (CMPS) was an efficient alternative to fluorocarbon surfactant, which was firstly synthesized via the esterification reaction using hydroxyl-containing polyether modified polysiloxane (HPMS) and maleic anhydride (MA) as raw materials. Then the surface activity, foam properties, micellar morphology of the CMPS and four hydrocarbon surfactants, as well as their mixture are evaluated by surface tension, Ross-Miles and cryo-transmission electron microscopy (Cryo-TEM). And the suitable key amount of CMPS and hydrocarbon surfactants added to the extinguishing agent were optimized by orthogonal tests. Moreover, the effect of foam stabilizer on the surface activity, foam property and fire extinguishing performance of fire extinguishing agent are also analyzed. Consequently, a new environmentally friendly fluorine-free fire extinguishing agent was prepared and the surface tension, the interfacial tension, foam properties, bubble morphology, spreading behavior and fire extinguishing mechanism were systematically characterized. The Cryo-TEM results illustrate that the intense interaction among silicone and hydrocarbon molecules exists in the the mixture of CMPS and hydrocarbon surfactants, which is produced through the change of micellar morphology, i.e. microscopic larger spheroidal micelles of CMPS transited to smaller spheroidal micelles and wormlike micelles in the mixed system. The surface tension of synthesized foam extinguishing agent is 22.3 mN/m and the spread coefficient is 0.9 mN/m, which has excellent surface activity and spreading property. The prepared foam extinguishing agent exhibits superior fire-fighting property, which can be attributed to the high surface activity, good foam property and superior foam stability. The research results of this paper lay a theoretical foundation for the research and development of efficient environmentally friendly foam extinguishing agent.

MRGPRX4 mediates phospho-drug-associated pruritus in a humanized mouse model.

The phosphate modification of drugs is a common chemical strategy to increase solubility and allow for parenteral administration. Unfortunately, phosphate modifications often elicit treatment- or dose-limiting pruritus through an unknown mechanism. Using unbiased high-throughput drug screens, we identified the Mas-related G protein-coupled receptor X4 (MRGPRX4), a primate-specific, sensory neuron receptor previously implicated in itch, as a potential target for phosphate-modified compounds. Using both Gq-mediated calcium mobilization and G protein-independent GPCR assays, we found that phosphate-modified compounds potently activate MRGPRX4. Furthermore, a humanized mouse model expressing MRGPRX4 in sensory neurons exhibited robust phosphomonoester prodrug-evoked itch. To characterize and confirm this interaction, we further determined the structure of MRGPRX4 in complex with a phosphate-modified drug through single-particle cryo-electron microscopy (cryo-EM) and identified critical amino acid residues responsible for the binding of the phosphate group. Together, these findings explain how phosphorylated drugs can elicit treatment-limiting itch and identify MRGPRX4 as a potential therapeutic target to suppress itch and to guide future drug design.

Structure and conformational variability of the HER2-trastuzumab-pertuzumab complex

Single particle analysis from cryogenic transmission electron microscopy (cryo-EM) is particularly attractive for complexes for which structure prediction remains intractable, such as antibody-antigen complexes. Here we obtain the detailed structure of a particularly difficult complex between human epidermal growth factor receptor 2 (HER2) and the antigen-binding fragments from two distinct therapeutic antibodies binding to distant parts of the flexible HER2, pertuzumab and trastuzumab (HTP). We highlight the strengths and limitations of current data processing software in dealing with various kinds of heterogeneities, particularly continuous conformational heterogeneity, and in describing the motions that can be extracted from our dataset. Our HTP structure provides a more detailed view than the one previously available for this ternary complex. This allowed us to pinpoint a previously overlooked loop in domain IV that may be involved both in binding of trastuzumab and in HER2 dimerization. This finding may contribute to explain the synergistic anticancer effect of the two antibodies. We further propose that the flexibility of the HTP complex, beyond the difficulties it causes for cryo-EM analysis, actually reflects regulation of HER2 signaling and its inhibition by therapeutic antibodies. Notably we obtain our best data with ultra-thin continuous carbon grids, showing that with current cameras their use to alleviate particle misdistribution is compatible with a protein complex of only 162 kDa. Perhaps most importantly, we provide here a dataset for such a smallish protein complex for further development of software accounting for continuous conformational heterogeneity in cryo-EM images.

Mechanisms of ligand recognition and activation of melanin-concentrating hormone receptors

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide that regulates food intake, energy balance, and other physiological functions by stimulating MCHR1 and MCHR2 receptors, both of which are class A G protein-coupled receptors. MCHR1 predominately couples to inhibitory G protein, Gi/o, and MCHR2 can only couple to Gq/11. Here we present cryo-electron microscopy structures of MCH-activated MCHR1 with Gi and MCH-activated MCHR2 with Gq at the global resolutions of 3.01 Å and 2.40 Å, respectively. These structures reveal that MCH adopts a consistent cysteine-mediated hairpin loop configuration when bound to both receptors. A central arginine from the LGRVY core motif between the two cysteines of MCH penetrates deeply into the transmembrane pocket, triggering receptor activation. Integrated with mutational and functional insights, our findings elucidate the molecular underpinnings of ligand recognition and MCH receptor activation and offer a structural foundation for targeted drug design.

Impact of distinct FG nucleoporin repeats on Nup98 self-association

Nucleoporins rich in phenylalanine/glycine (FG) residues form the permeability barrier within the nuclear pore complex and are implicated in several pathological cellular processes, including oncogenic fusion condensates. The self-association of FG-repeat proteins and interactions between FG-repeats play a critical role in these activities by forming hydrogel-like structures. Here we show that mutation of specific FG repeats of Nup98 can strongly decrease the protein’s self-association capabilities. We further present a cryo-electron microscopy structure of a Nup98 peptide fibril with higher stability per residue compared with previous Nup98 fibril structures. The high-resolution structure reveals zipper-like hydrophobic patches which contain a GLFG motif and are less compatible for binding to nuclear transport receptors. The identified distinct molecular properties of different regions of the nucleoporin may contribute to spatial variations in the self-association of FG-repeats, potentially influencing transport processes through the nuclear pore.

Engineered Phenylalanine Ammonia-Lyases for the Enantioselective Synthesis of Aspartic Acid Derivatives.

Biocatalytic hydroamination of alkenes is an efficient and selective method to synthesize natural and unnatural amino acids. Phenylalanine ammonia-lyases (PALs) have been previously engineered to access a range of substituted phenylalanines and heteroarylalanines, but their substrate scope remains limited, typically including only arylacrylic acids. Moreover, the enantioselectivity in the hydroamination of electron-deficient substrates is often poor. Here, we report the structure-based engineering of PAL from Planctomycesbrasiliensis (PbPAL), enabling preparative-scale enantioselective hydroaminations of previously inaccessible yet synthetically useful substrates, such as amide- and ester-containing fumaric acid derivatives. Through the elucidation of cryo-electron microscopy (cryo-EM) PbPAL structure and screening of the structure-based mutagenesis library, we identified the key active site residue L205 as pivotal for dramatically enhancing the enantioselectivity of hydroamination reactions involving electron-deficient substrates. Our engineered PALs demonstrated exclusive a-regioselectivity, high enantioselectivity, and broad substrate scope. The potential utility of the developed biocatalysts was further demonstrated by a preparative-scale hydroamination yielding tert-butyl protected l-aspartic acid, widely used as intermediate in peptide solid-phase synthesis.

Additive-Driven Nanoscale Architecture of Solid Electrolyte Interphase Revealed by Cryogenic Transmission Electron Microscopy.

In Li metal batteries (LMBs), which boast the highest theoretical capacity, the chemical structure of the solid electrolyte interphase (SEI) serves as the key component that governs the growth of reactive Li. Various types of additives have been developed for electrolyte optimization, representing one of the most effective strategies to enhance the SEI properties for stable Li plating. However, as advanced electrolyte systems become more chemically complicated, the use of additives is empirically optimized. Indeed, their role in SEI formation and the resulting cycle life of LMBs are not well-understood. In this study, we employed cryogenic transmission electron microscopy combined with Raman spectroscopy, theoretical studies including molecular dynamics (MD) simulations and density functional theory (DFT) calculations, and electrochemical measurements to explore the nanoscale architecture of SEI modified by the most representative additives, lithium nitrate (LiNO3) and vinylene carbonate (VC), applied in a localized high-concentration electrolyte. We found that LiNO3 and VC play distinct roles in forming the SEI, governing the solvation structure, and influencing the kinetics of electrochemical reduction. Their collaboration leads to the desired SEI, ensuring prolonged cycle performance for LMBs. Moreover, we propose mechanisms for different Li growth and cycling behaviors that are determined by the physicochemical properties of SEI, such as uniformity, elasticity, and ionic conductivity. Our findings provide critical insights into the appropriate use of additives, particularly regarding their chemical compatibility.